Cyanogenic clovers invest more in rhizobia but do not affect rhizobium evolution

Nitrogen governs the interactions between leguminous plants and their nitrogen-fixing resource mutualists, rhizobia. While most work has focused on how soil nitrogen availability shapes the ecology and evolution of the legume-rhizobium mutualism, the nitrogen demands of plants may also influence the mutualism. Plants differ in nitrogen demand, in part because of variation in specialized metabolite production, particularly in species that produce nitrogen-based defense compounds such as cyanogenic glycosides. Plants with high nitrogen demand might be expected to be more reliant on rhizobia, potentially investing more in rhizobia and exerting stronger preferences for high-quality mutualists. Here, we use white clover (Trifolium repens), which is polymorphic for cyanogenesis, from two biparental F3 mapping populations to examine whether nitrogen-based chemical defenses affect plant investment in the mutualism or rhizobium evolution. By inoculating cyanogenic and acyanogenic clover lines with a single strain of Rhizobium leguminosarum, we found that cyanogenic clovers produced larger root nodules than acyanogenic clover, suggesting that cyanogensis is associated with increased investment in rhizobia. Greater investment in rhizobia could select for more cooperative mutualists if accompanied by partner choice or sanctions allowing plants to preferentially reward more cooperative rhizobia. We conditioned soils with cyanogenic and acyanogenic clover lines for five plant growth cycles and compared the partner quality of rhizobia isolated from these soils. Rhizobia evolved in association with cyanogenic versus acyanogenic clover did not differ in quality. Overall, our results show that N-based anti-herbivore defense traits can influence plant investment in resource mutualists but suggest that differences in investment do not necessarily translate into long-term evolutionary changes in mutualist quality.